Abstract: Disclosed is a method for constructing a nucleus-implanted egg, a parthenogenetic embryo and for producing a parthenogenetic mammal each having 2 haploid genome sets originating in mammalian ova, and provides methods of constructing a nucleus-implanted egg having a haploid genome set derived from primitive ovarian follicle egg (ng ovum) and a haploid genome set from MII phase (second meiosis metaphase) egg (fg ovum), a parthenogenetic embryo and a parthenogenetic mammal, including steps (1) introducing ng ovum into a nucleus-deleted deleted germinal vesicle stage (GV) egg, developing the obtained egg to MII phase by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and (2) extracting MII phase chromosome from the first nucleus-implanted egg and introducing it into other fg ovum to prepare a second nucleus-implanted egg, wherein a ng or fg ovum from which an imprinted gene undergoing epigenetic modification during sperm generation is used.

Abstract: The present invention relates to a genetically modified pig as a model for studying breast cancer, mitochondria related protein folding disorders and/or epidermolysis bullosa simplex. The modified pig model displays one or more phenotypes associated with any of said disorders. Disclosed is also a modified pig comprising a modified endogeneous BRCA1 and/or BRCA 2 gene, and/or a modified ornithine transcarbamylase gene, and/or a modified Keratin 14 gene and/or a transcriptional or translational product or part thereof. The invention further relates to methods for producing the modified pig; and methods for evaluating the effect of a therapeutical treatment of breast cancer, mitochondria related protein folding disorders and/or epidermolysis bullosa simplex; methods for screening the efficacy of a pharmaceutical composition; and a method for treatment of a human being suffering from breast cancer, mitochondria related protein folding disorders and/or epidermolysis bullosa simplex are disclosed.

Abstract: Disclosed are a cloned pig expressing green fluorescent protein (GFP) and a cloned pig having a 1,3-galactosyltransferase (GT) gene knocked out. Also, the present invention discloses methods of producing such cloned pigs, comprising the steps of establishing a somatic cell line; preparing a GFP-transfected or GT gene knock-out nuclear donor cell; producing a transgenic nuclear transfer embryo using the nuclear donor cell and a recipient oocyte; and transplanting the transgenic nuclear transfer embryo into a surrogate mother pig. The cloned pig expressing GFP of the present invention is useful for large-scale production of an animal disease model, and the GT gene knock-out cloned pig can be used as a organ donor allowing xenotransplantation in humans without hyperacute immune rejection.

Abstract: The invention relates to a gene (cDNA) encoding a bovine myostatin protein. The nucleic acid coding sequence is identified as SEQ ID NO:1 and the protein sequence is identified as SEQ ID NO:2. A mutant gene (SEQ ID NO:3) in which the coding sequence lacks an 11-base pair consecutive sequence (SEQ ID NO:11) of the sequence encoding bovine protein having myostatin has been sequenced. It has been shown that cattle of the Belgian Blue breed homozygous for the mutant gene lacking myostatin activity are double-muscled. A method for determining the presence of muscular hyperplasia in a mammal is described. The method includes obtaining a sample of material containing DNA from the mammal and ascertaining whether a sequence of the DNA encoding (a) a protein having the biological activity of myostatin, is present and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a), is present.

Abstract: An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapies and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

Abstract: The present invention provides a method of producing a cloned mammal, which uses a mammalian natural killer T cell as a donor cell, a cloned mammal obtained by the method, a method of obtaining an ES cell from the embryo of the cloned animal and an ES cell obtained by the method.

Abstract: Compositions and methods are provided for the efficient and reproducible generation of clone animals of all developmental stages. Also provided are methods of use of the same in reproductive and therapeutic cloning protocols.

Abstract: This invention relates to the isolation and propagation of pluripotent cells isolated from the mammalian late epiblast layer, termed Epiblast Stem Cells' (EpiSCs). These cells are useful in a range of applications, including the generation of transgenic animal species.

Abstract: Disclosed is a method for constructing a nucleus-implanted egg, a parthenogenetic embryo and for producing a parthenogenetic mammal each having 2 haploid genome sets originating in mammalian ova, and provides methods of constructing a nucleus-implanted egg having a haploid genome set derived from primitive ovarian follicle egg (ng ovum) and a haploid genome set from MII phase (second meiosis metaphase) egg (fg ovum), a parthenogenetic embryo and a parthenogenetic mammal, including steps (1) introducing ng ovum into a nucleus-deleted deleted germinal vesicle stage (GV) egg, developing the obtained egg to MII phase by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and (2) extracting MII phase chromosome from the first nucleus-implanted egg and introducing it into other fg ovum to prepare a second nucleus-implanted egg, wherein a ng or fg ovum from which an imprinted gene undergoing epigenetic modification during sperm generation is used.

Abstract: Transgenic and cloned ungulates and particularly cloned cattle are disclosed, wherein such cattle contain a deletion or disruption of the prion gene locus and do not express functional prion protein, and are not susceptible to prion-related diseases such as bovine spongiform encephalopy or Mad Cow Disease.

Abstract: The invention is directed in part to totipotent cells that have one or more artificial chromosomes; processes for producing such cells; processes for using such cells (e.g., nuclear transfer); transgenic embryos and transgenic animals cloned from such cells; and processes for producing such embryos and animals.

Abstract: The present invention relates to a method for producing a chimeric non-human animal expressing a desired protein, and a chimeric non-human animal or an offspring thereof expressing a desired protein. The present invention also relates to a method for analyzing the functions of a desired protein or a gene encoding the protein by comparing the phenotype of the above chimeric non-human animal with that of a corresponding wild-type animal.

Abstract: The present invention provides a GLAST knockout mouse lacking the function of an endogenous glutamate transporter GLAST gene, which shows: 1) an intraocular pressure within the normal range; and 2) a reduction in the number of cells in the retinal ganglions when compared with a wild-type normal mouse. Owing to the ocular properties, this knockout mouse is useful as a model for normal tension glaucoma. By using this knockout mouse, a compound useful for the treatment of normal tension glaucoma can be screened.

Abstract: Provided herein are mitochondrial-nuclear exchanged cells and animals comprising mitochondrial DNA (mtDNA) from one subject and nuclear DNA (nDNA) from a different subject. Methods for producing a mitochondrial-nuclear exchanged animal and animals made by the methods are provided. Also provided are methods of screening for agents useful for treating a disease or disorder using mitochondrial-nuclear exchanged animals or cells, tissues or organs thereof.

Abstract: A method of assessing the viability of a cell comprises incubating the cell in a culture medium. The culture medium includes a plurality of amino acids and the change in concentration in the medium of at least one amino acid is determined.

Abstract: The present invention relates to a method of producing an ungulate having both copies of the IgM heavy chain (mu) rag-1 and/or rag-2 gene eliminated from its genome. Animals which have IgM, rag-1 and/or rag-2 eliminated from their genome are unable to conduct the gene rearrangements that are necessary to generate the antigen receptors of B- or T-lymphocytes, and therefore will not develop native B- or T-cells. Because they are unable to produce B- and T-lymphocytes, these IgM, rag-1, or rag-2 ungulates cannot reject human hematopoietic stem cell preparations, and B- and T-lymphocytes which develop therefrom. Therefore, the present invention also involves injecting into IgM, rag-1, and/or rag-2 deficient ungulates, in utero or shortly after birth, human B- and T-lymphocytes whose immune systems produce human immunoglobulin that can be processed for therapeutic uses in humans.

Abstract: A nuclear transfer embryo is formed by destabilizing microtubules of an oocyte, whereby essentially all endogenous chromatin collects at a second polar body during meiosis of an oocyte. The oocyte is fused with the nucleus of a donor somatic cell of the same species of said oocyte prior to cessation of extrusion of the second polar body from the oocyte, thereby forming the nuclear transfer embryo. In one embodiment, the nuclear transfer embryo is employed to impregnate an animal, such as a mammal. In another embodiment, the donor nucleus is transgenic.

Abstract: A cloned animal is produced by demecolcine treatment characterized by culturing the nuclear transfer embryo in vitro and transferring the embryo in vivo. This significantly improves in vitro development of somatic cell nuclear transfer embryos and maintenance of pregnancy from transfer of transfer somatic cell nuclear transfer embryos in a surrogate mother up to delivery. A nuclear transfer embryo of a non-primate mammal made by enucleating a recipient oocyte; injecting a nuclear donor cell into the enucleated oocyte; fusing together the injected enucleated oocyte with the injected nuclear donor cell to form a fused oocyte; activating the fused oocyte; and treating the activated oocyte with demecolcine to form the nuclear transfer embryo.

Abstract: An object of the invention is to effectively prepare a fish embryo with a correct chromosomal ploidy by nuclear transplantation in which an exogenous fish nucleus is transplanted in a cytoplasmic recipient. For this object, the invention comprises a step of preparing a fish embryo by transplanting a fish cell nucleus to an unfertilized egg. The step of preparing a fish embryo comprises a step of imposing physical and/or chemical stress to the unfertilized egg after activation. By imposing such stress, the stage of haplosis in a female nucleus which happens at the early stage of a series of developmental steps occurring in an unfertilized egg is suppressed and the correct ploidy of an obtained embryo is at least secured.

Abstract: This invention relates to the reprogramming of animal cells and animal cell nuclei by nuclear addition. The invention also relates to the generation of animal cells, cell lines, tissues, organs, embryos and non-human animals by methods involving nuclear addition.

Abstract: The invention provides a transgenic Mustelidae in which a gene associated with a human disease or condition comprises a targeted genetic modification, and uses thereof. Also provided is a method to cryopreserve Mustelidae embryos or cells, and to enhance the number of live offspring from cryopreserved Mustelidae embryos.

Abstract: The present invention pertains to methods for cloning animals. In particular, the invention includes methods of cloning an animal by combining a genome from an activated donor cell with an activated enucleated oocyte to thereby obtain a nuclear transfer embryo, and impregnating an animal with the nuclear transfer embryo in conditions suitable for gestation of a cloned animal. The invention further relates to methods of chemically enucleating an oocyte having a meiotic spindle apparatus by exposing the oocyte with a compound that destabilizes the meiotic spindle apparatus.

Abstract: The present invention provides transgenic, large non-human animal models of diseases and conditions, as well as methods of using such animal models in the identification and characterization of therapies for the diseases and conditions.

Abstract: The invention relates to the genetic manipulation of non-human animals. More particularly, the invention relates to genetic manipulation of non-human animals to be used for xenotransplantation. The invention provides viable gene knockout swine including swine in which the ?(1,3)-galactosyltransferase gene has been disrupted, methods for making such swine, and methods of using the tissues and organs of such swine for xenotransplantation.

Abstract: The present invention provides methods of producing a clone non-human mammalian nuclear transfer (NT) embryo and methods for producing a cloned non-human mammal. Embodiments of the methods include introducing doner genetic material into a metaphase I oocyte; introducing donor genetic material into a non-enucleated oocyte; introducing donor genetic material obtained from a donor cell that is a metaphase into an oocyte; introducing donor genetic material into an oocyte, and naturally activating the oocyte or the NT embryo; and introducing donor genetic material obtained from a donor cell that is at late G1 phase into anoocyte.

Abstract: The observed over-expression of Sp1 target genes has inspired inventors to formulate a specific strategy for correcting many gene expression defects, and thus improve clone development. The invention is based on the belief that manipulating Sp1 activity can improve cloning. Inventors believe that cloning is inefficient in large part because of the continued expression of Sp1 target genes in the early cloned embryos, which causes clones to be very unlike normal embryos, and so makes them very unhealthy. Inventors propose that if over-expression of Sp1 target genes is prevented in early stage clones, this would greatly improve cloning efficiency by making the cloned embryos healthy again.

Abstract: The present invention provides systems that allow reliable multiplexed transformation of Drosophila embryos. The present invention provides methods and reagents that allow preparation of injection-quality nucleic acid samples and that allow simultaneous preparation of multiple such samples. The present invention provides systems for simultaneous processing of multiple injected embryos. The present invention provides methods for transformation of Drosophila embryos involving use of virginator strains that can be used to increase the efficiency of setting up the crosses needed to produce the eggs for the injections and for the crosses needed to screen for transformants.

Abstract: A nuclear transfer method is provided wherein nuclear DNA in whole or part is injected into enucleated oocytes. The method is suitable for different donor cells, and preferably ES cells.

Abstract: A method is described for the production of an animal (offspring) by the process of nuclear transfer. The animal may be produced using donor genetic material in cells taken directly from any stage of an animal, such as an embryo, foetus or adult or from cell cultures established from material from any stage of an animal, such as embryonic, foetal or adult material. The process may be used to introduce genetic modifications into the resultant offspring by genetic manipulation and selection of the cells to act as nuclear donors prior to embryo reconstruction. The present invention provides a method of reconstituting an animal embryo, the process comprising transferring a nucleus into a first oocyte followed by removing and transferring the nucleus from the oocyte to a further oocyte or to an enucleated fertilized zygote.

Abstract: The present invention discloses a process for obtaining somatic cell derived embryonic stem cells (encoded by reprogrammed somatic cell nuclei), ES cell-like cells or other types of embryo-derived stem cells by nuclear transplantation, and a process for inducing said stem cells into various differentiated cell types.

Abstract: A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Abstract: Transgenic mice that have been engineered to be deficient in the gene encoding the neuromedin receptor subtype 1 gene. Such mice are useful in screening for receptor subtype-specific agonists and antagonists.

Abstract: The present invention provides compositions and methods for increasing the success of assisted reproductive technology (ART). Specifically, the inventions described herein increase the survival rate of manipulated embryos for increasing post implantation numbers of viable offspring. In particular, the present invention provides for compositions and methods for allowing further embryonic development and increasing rates of embryonic maturation, such as increasing cleavage rate, TE numbers, and blastocyte formation of in vitro fertilized and nuclear transfer embryos in media comprising follistatin, thereby providing for increased survival of fertilized and manipulated embryos leading to increased numbers of live offspring from in vitro fertilized and implanted nuclear transfer embryos. Further provided are diagnostic kits for determining transplantation potential.

Abstract: The present invention relates to a non-human transgenic animal, particularly a knock in mouse, whose genome comprises a heterologous, chimeric CTLA4 gene. The chimeric CTLA4 gene comprises exon 2 of the human CTLA4 gene, exon 1 and exon 4 of the non-human animal, and exon 3 of the CTLA4 gene of the non-human animal, or preferably, exon 3 of the human CTLA4 gene. The invention also relates to methods by which the transgenic mice are used to screen for monoclonal antibodies or other molecules that enhance immunity to tumors and infectious agents by interacting with the human CTLA4 receptor. The transgenic mice of the present invention are also useful for screening for monoclonal antibodies or other molecules that inhibit autoimmunity and transplant rejection.

Abstract: It is intended to provide a method for improving a development rate in a somatic nuclear transplantation technique or an artificial insemination technique using a spermatid. The method is a method for producing a nuclear-transplanted egg comprising the steps of transplanting a nucleus of a donor cell into an egg, and treating the nuclear-transplanted egg with an anti-methylating agent.

Abstract: The present invention provides porcine CMP-N-Acetylneuraminic-Acid Hydroxylase (CMP-Neu5Ac hydroxylase) protein, cDNA, and genomic DNA regulatory sequences. Furthermore, the present invention includes porcine animals, tissues, and organs, as well as cells and cell lines derived from such animals, tissues, and organs, which lack expression of functional CMP-Neu5Ac hydroxylase. Such animals, tissues, organs, and cells can be used in research and in medical therapy, including in xenotransplantation, and in industrial livestock farming operations.

Abstract: The present invention is to provide a method for constructing a nucleus-implanted egg, a parthenogenetic embryo and for producing a parthenogenetic mammal each having 2 haploid genome sets originating in mammarian ova, and provides methods of constructing a nucleus-implanted egg having a haploid genome set derived from ng ovum and a haploid genome set from fg ovum, a parthenogenetic embryo and a parthenogenetic mammal, which includes the steps of (1) introducing a primitive ovarian follicle egg (ng ovum) into a nucleus-deleted egg in a germinal vesicle stage (GV stage egg) and then developing them to MII phase (second meiosis metaphase) by in vitro maturing and culturing to prepare a first nucleus-implanted egg, and (2) extracting MII phase chromosome from said first nucleus-implanted egg and introducing it into other MII phase egg (fg ovum) to prepare a second nucleus-implanted egg, wherein ovum from which an imprinted gene that undergoes gene modification posteriori during the generation of sperm is deleted

Abstract: The invention relates to a method for cloning in the rat by nuclear transfer. The invention further relates to the rats obtained thus, in the foetal or adult state, as well as use thereof for the production of molecules of interest or as study models.

Abstract: The present invention relates to a method for producing a modified foreign chromosome(s) or a fragment(s) thereof, which comprises the steps of: (a) preparing a microcell comprising a foreign chromosome(s) or a fragment(s) thereof, and transferring said foreign chromosome(s) or a fragment(s) into a cell with high homologous recombination efficiency through its fusion with said microcell; (b) in said cell with high homologous recombination efficiency, inserting a targeting vector by homologous recombination into a desired site of said foreign chromosome(s) or a fragment(s) thereof, and/or a desired site of a chromosome(s) derived from said cell with high homologous recombination efficiency, thereby marking said desired site; and (c) in said cell with high homologous recombination efficiency, causing deletion and/or translocation to occur at the marked site of said foreign chromosome(s) or a fragment(s) thereof.

Abstract: The present invention is directed to various methodologies to make NT a practical procedure for animals, specifically, primates including human and non-human primates. Furthermore, the methods and molecular components provided by the present invention provide a practical means for producing embryos with desired characteristics. In a specific embodiment, the methodology of the present invention comprises introducing nuclei having desired characteristics along with one or more molecular components into an enucleated egg, thus creating a nuclear transfer construct, culturing the egg to produce a viable embryo, transferring the embryo to the oviducts of a female, and producing a cloned animal.

Abstract: Provided is a method of effectively producing a cloned animal by demecolcine treatment. A conventional method of producing a nuclear transfer embryo of an animal further includes demecolcine treatment after activation of a fused oocyte. Moreover, the method of producing a cloned animal is characterized by culturing the nuclear transfer embryo in vitro and transferring the embryo in vivo. This method significantly improves in vitro development of somatic cell nuclear transfer embryos and maintenance of pregnancy from transfer of somatic cell nuclear transfer embryos in a surrogate mother up to delivery.

Abstract: Provided is a method for producing a cloned dog by enucleating an oocyte of a dog to produce an enucleated oocyte, transferring a somatic cell of the dog into the enucleated oocyte, carrying out electrofusion under optimized conditions to produce a nuclear transfer embryo, and transferring the nuclear transfer embryo into its surrogate mother. The method has the effect of producing a cloned dog with high efficiency, and thus can contribute to the development of studies in veterinary medicine, anthropology and medical science, such as the propagation of superior species, xenotransplantation and diseased animal models. In addition, the present invention has the effect of exactly researching production properties of the cloned dog by producing a female cloned dog for the first time, rather than the conventional male cloned dog.

Abstract: Methods for the production of nuclear transfer embryos, nuclear transfer embryos and animals derived therefrom are described. The method generally comprises at least the steps of: providing at least one enucleated recipient cell; providing at least one donor cell or nucleus; providing a fusion media which is substantially free of calcium; placing said at least one enculeated recipient cell and at least one donor cell or nucleus in contact with one another to form couplets; and, fusing via electrofusion in said fusion media said at least one recipient cell with at least one donor cell or nucleus to form a nuclear transfer embryo.

Abstract: The present invention is directed to various methodologies to make NT a practical procedure for animals, specifically, primates including human and nonhuman primates. Furthermore, the methods and molecular components provided by the present invention provide a practical means for producing embryos with desired characteristics. In a specific embodiment, we methodology of the present invention comprises introducing nuclei having desired characteristics along with one or more molecular components into an egg, culturing the egg to produce a viable embryo, transferring the embryo to the oviducts of a female, and producing a cloned animal.

Abstract: A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Abstract: The invention provides cloned transgenic ungulates (e.g., bovines) in which prion protein activity is reduced by one or more genetically engineered mutations. Desirably, these transgenic bovines are also genetically modified to express xenogenous (e.g., human) antibodies. Because of their resistance to prion-related diseases such as bovine spongiform encephalopy (also known as mad cow disease), these bovines are a safer source of human antibodies for pharmaceutical uses and a safer source of agricultural products.

Abstract: Disclosed herein are methods of generating a recipient cytoplast for nuclear cloning with improved developmental capacity. These methods involve the in vitro maturation of an oocyte from a non-human mammal to the pre-MII stage and treatment of the oocyte with a microtubule-inhibiting agent prior to enucleation. The oocyte used to generate the recipient cytoplast can be obtained from a pre-adult or adult non-human mammal. The methods further provide for utilizing the generated recipient cytoplast for nuclear transfer, such as deriving a nuclear transfer embryo or offspring.

Abstract: The invention relates to a method for cloning in the rat by nuclear transfer. The invention further relates to the rats obtained thus, in the foetal or adult state, as well as use thereof for the production of molecules of interest or as study models.

Abstract: Transgenic mice that have been engineered to be deficient in the gene encoding the neuromedin receptor subtype 1 gene. Such mice are useful in screening for receptor subtype-specific agonists and antagonists.

Abstract: The present invention relates to methods for producing transgenic animals, particularly transgenic rats, using retroviral constructs engineered to carry the transgene(s) of interest.